AU652946B2 - The pharmacological use of certain cystine derivatives - Google Patents

Abstract

The invention concerns pharmaceutical preparations for stimulation of the immune system which comprise a cystine derivative as active ingredient.

Description

OPI DATE 31/12/91 APPLN. ID 80941 91 REATY (PCT) INTERNAl AOJP DATE 30/01/92 (51) International Patent Classification 5 A61K 31/195, 31/22, 31/225 C07C 323/59 PCT NUMBER PCT/SE91/00388 (11) International Publication Number: WO 91/18594 Al (43) International Publication Date: 12 December 1991 (12.12.91) (21) International Application Number: (22) International Filing Date: PCT/SE91/00388 3 June 1991 (03.06.91) Priority data: 9002067-8 9002275-7 8 June 1990 (08.06.90) 28 June 1990 (28.06.90) (71) Applicant (for all designated States except US): AKTIEBO- LAGET ASTRA [SE/SE]; S-151 85 S6dertflje (SE).

(72) Inventors; and Inventors/Applicants (for US only) ANDERSSON, Carl- Magnus, Alexander [SE/SE]; Filippavlgen 6B, S-222 41 Lund BERGSTRAND, Sten, HAkan, Axel [SE/ SE]; S. Villavigen 2, S-230 50 Bjlrred HALL- BERG, Anders, Rudolf [SE/SE]; SAngarevigen 8D, S- 223 71 Lund SARNSTRAND, Bengt, Olof [SE/ SE]; Leifs vig 28, S-237 00 Bjlrred TUNEK, Per, Anuers, Sigvard [SE/SE]; Stadiongatan 53A, S-217 62 MalmB (SE).

(74) Agents: DANIELSSON, Sten et al.; AB Astra, Patent Department, S-151 85 Sodertalje (SE).

(81) Designated States: AT, AT (European patent), AU, BB, BE (European patent), BF (OAPI patent), BG, BJ (OAPI patent), BR, CA, CF (OAPI patent), CG (OAPI patent), CH, CH (European patent), CI (OAPI patent), CM (OAPI patent), DE, DE (European patent), DK, DK (European patent), ES, ES (European patent), FI, FR (European patent), GA (OAPI patent), GB, GB (European patent), GN (OAPI patent), GR (European patent), HU, IT (European patent), JP, KP, KR, LK, LU, LU (European patent), MC, MG, ML (OAPI patent), MR (OAPI patent), MW, NL, NL (European patent), NO, PL, RO, SD, SE, SE (European patent), SN (OAPI patent), SU, TD (OAPI patent), TG (OAPI patent), US.

Published With international search report.

46 (54) Title: THE PHARMACOLOGICAL USE OF CERTAIN CYSTINE DERIVATIVES (57) Abstract A method for the treatment of diseases due to defects in the immune system using certain cystine derivatives and a pharmaceutical preparation comprising these derivatives.

WO 91/18594 P~r/SE990388 1 THE PHARMACOLOGICAL USE OF CERTAIN CYSTINE DERIVATIVES Field of the Invention The present invention relates to a new medical use of N,N'-diacetylcystine, N,N'-dibutyrylcystine, N,N'diisovalerylcystine, N, N'-dicaprylylcystine, N,N'diacetylcystine dimethyl ester, N,N'-diacetylcystine diethyl ester and N,N'-diisovalerylcystine dimethyl ester in racemic forms or in the form of optical D or L isomers.

In particular the invention relates to the use of the abovementioned compounds for the preparation of medicaments with immunomodulating action, particularly immunostimulating action.

Background of the Invention N-Acetyl-L-cysteine is a compound widely used for treating chronic obstructive airway diseases/chronic bronchitis (for further references see Multicentre Study Group.

Long-term oral acetylcysteine in chronic bronchitis. A double-blind controlled study. Eur. J. Respir. Dis. 1980, 61 (suppl. 111), 93-108; Boman, Backer, Larsson, Melander, and WAhlander, L. Oral acetylcysteine reduces exacerbation rate in chronic bronchitis. Report of a trial organized by the Swedish Society for Pulmonary Disease. Eur. J. Respir. Dis. 1983, 64, 405-415; and British Thoracic Society Research Committee. Oral Nacetylcysteine and exacerbation rates in patients with chronic bronchitis and severe airway obstruction. Thorax 1985, 40, 832-835). The mechanism of action of the compound is not disclosed; its effect has been attributed to mucolytic properties (see Multicentre Study Group.

WO 91/18594 PC'F/SE91/00388 2 Long-term oral acetylcysteine in chronic bronchitis. A double-blind controlled study. Eur. J. Respir. Dis. 1980, 61 (suppl. 111), 93-108; Boman, Backer, Larsson, Melander, and WAhlander, L. Oral acetylcysteine reduces exacerbation rate in chronic bronchitis. Report of a trial organized by the Swedish Society for Pulmonary Disease. Eur. J. Respir. Dis. 1983, 64, 405-415; and British Thoracic Society Research Committee. Oral Nacetylcysteine and exacerbation rates in patients with chronic bronchitis and severe airway obstruction. Thorax 1985, 40, 832-835), antioxidant properties (see Aruoma, Halliwell, Hoey, and Butler, J. Free Radical Biol. Med. 1989, 6, 593-597), and also immunomodulating properties (see Bergstrand, Bj6rnson, Eklund, Hernbrand, Eklund, Larsson, K., Linden and Nilsson, A. Stimuli-induced superoxide radical generation in vitro by human alveolar macrophages from smokers: Modulation by N-Acetylcysteine treatment in vivo. J. Free Radicals Biol. Med. 2, 1986, 119-127).

The present invention deals with the disulfide of Nacetylcysteine, that is N,N'-diacetylcystine (in the following referred to as DiNAC), i.e. the compound of the formula:

COOH

S-CH

2

-CH-NH-COCH

3

S-CH

2

-CH-NH-COCH

3 1

COOH

N,N' dibuty:ylcystine (in the following referred to as diBUT), i.e. the compound of the formula: WO 91/18594 PCr/SE91/00388 3 cOO1H S-CH 2 -CH-NH-COCH 2 CH 2 CH 3

S-CH

2 CH-NH-COCH 2 CH 2 CH 3

COON

N,N'-diisovalerylcystile (in the following reffered to as divAL), i.e. the compound of the formula COON

CH

3 I I

S-CH

2 -CH-NH-COCH 2 CHCH3 1 S-CH 2 -CH-NH-COCH 2

CHCH

3 I I COOH CH 3 N,NI-dicaprylylcystine (in the following referred to as diCAP), i.e. the compound of the formula

COON

S-CH 2 CH-NH-CO-(CH 2 6 CH 3 S-H2-C -H C (C 3

COON

N,NI-diacetylcystine dimethyl ester (in the following referred to as diI~eNAC), i.e. the compound of the formula: WO 91/18594 PCT/SE9I /00388

COOCH,

I-H C-N-OH

S-CH,-CH-NH-COCH,

COOCH,

N,N'-diacetylcystine diethyl ester (in the following referred to as diEtNAC), i.e. the compound of the formula:

COOCH,

S-CH,-CH-NII-COCH,

S-CH,-CH-NH-COCH,

1 COOC 2

H,

and N,N'-diisovalerylcystine dimethyl ester (in the following referred to as diMeVAL), i.e. the compound of the formula:

COOCH

3

CH,

S-CH,-CH-NH-COCH,-CHCH,

S-CH,-CH-NII-COCH,-CHCH,

COOCH, CH, The invention deals with the above mentioned compounds in racemic form as well as the isomeric D and L forms of the compounds. Of particular interest are the compounds having the L configuration, particularly interesting is N,N'diacetyl-L-cystine.

The invention also deals with the compounds in the form of their physiologically acceptable salts such as the salts WO 91/18594 P(3r/SE91/00388 of sodium, potassium, ammonium, calcium or magnesium.

Also included are salts of the compounds diNAC, diBUT, diVAL and diCAP with pharmaceutically acceptable organic bases.

The above mentioned compounds have previously been described in the patent literature as well as in the scientific literature. DiNAC in the following publications: US 4827016; EP 300100; US 4724239; US 4708965; DE 2326444; Wilson, and Nicholson, J.K.

Analysis of thiols and disulfides in Sulphur-containing drugs and related organic compounds. Chemistry, Biochemistry an Toxicology (ed L.A. Damani) Vol. 2A.

Analytical, biochemical and toxicological aspects of sulphur xenobiochemistry. Ellis Horwood Series in Biochemical Pharmacology (Halstred Press: a division of John Wiley Sons) Chichester 1989, p. 45; and Sj6din K., Nilsson Hallberg, and Tunek, A. Metabolism of N- Acetyl-L-cysteine. Some structural reguirements for the deacetylation and consequences for the oral biovailability. Biochem. Pharmacol. 1989, 38, 3981-3985).

In US 4827016 the compound is claimed to be effective for topical treatment of dermal inflammations which are induced and propagated by leukotrienes.

The remaining compounds have also been described in the literature. (See for instance, for diMeNAC: Bowman, W.R.

Richardson, G.D. Tetrahedron Lett. 1981, 22, 1551-1554; for diEtNAC: Damico, R.A. Boggs, R.W. US 3952115 (1976); for diVAL, diMeVAL: Martin, T.A. J. Med. Chem 1969, 12, 950-953), for diCAP: FR 8205 M, for diBUT: FR 2503151).

Nothing is reported or generally known concerning the pharmacological and/or therapeutic properties of these compounds with respect to immunological systems or inflammatory diseases of the lung such as chronic WO 91/18594 PCT/SE91/0388 6 bronchitis.

Disclosure of the Invention It has unexpectedly been found that the hereinbefore mentioned compounds diNAC, diBUT, diVAL, diCAP, diMeNAC, diEtNAC and diMeVAL in an experimental animal model for assessing a T-cell reactivity in vivo, i.e. the delayed type hypersensitivity (DTH) reaction in the mouse ear, are highly potent and efficient immunostimulating agents, some being in the order of 100-1000 times more effective than the thiol NAC. Thus, in this model the compounds are highly effective immunostimulators with a potency and efficacy superior or equal to known immunostimulants such as diethyl dithiocarbamate (DTC) or hydroxyethyl disulfide (HEDS; see St Georgiev, V. New synthetic immunomodulating agents. Trends in Pharmacological Science 1988, 446-451).

Therefore, the compounds DiNAC, diBUT, diVAL, diCAP, diMeNAC, diEtNAC, diMeVAL and their D and L optical isomers may be used for treatment of diseases where a defect in the immune system and/or an ineffective host defence is at hand or can be suspected.

Examples of such diseases are chronic bronchitis and other inflammatory diseases of the airways such as asthma and rhinitis but also certain forms of autoimmune diseases like diabetes and rheumatoid arthritis and/or various malignant diseases. HIV infection or AIDS may be treated with the compounds. Also atherosclerotic disease may be treated with the compounds.

Effective amounts of the compounds diNAC, diBUT, diVAL, diCAP, diMeNAC, diEtNAC, diMeVAL and their D and L optical isomers for use in the treatment of the above mentioned diseases are in the range 0.5-500 mg, preferably 5-50 mg, daily dose, which can be expressed as 0.007-0.7 mg/kg, preferably 0.07-0.7 mg/kg patient body weight per day, applied in dosage unit form preparations formulated to contain 1-10 mg of active substance per unit, as shown in Examples 1-5 hereinafter.

Synthesis of compounds The compounds diNAC, diBUT, diVAL and diCAP may be prepared, for example, from L-cystine via acylation (see US 4827016; EP 300100; US 4724239; US 4708965; DE 2326444; Marshall, Winitz, Birnbaum, S.M. and Greenstein, J.P. J. Am. Chem. Soc. 1957, 79, 4538-4544; and Cecil, R.

McPhee, J.B. Biochem. J. 19-7, 66, 538-543) or through oxidative dimerization of the appropriate acylcysteines (see Snow, Finley, J.W. Friedman, M. Biochem.

Biophys. Res. Commun. 1975, 64, 441-447).

The esters diMeNAC, diEtNAC and diMeVAL may be synthesized analogously, i.e. by acylation of the cystine methyl or ethyl esters as appropriate or by oxidative dimerisation of the respective N-acetyl cystine methyl or ethyl esters or N-isovalerylcysteine methyl ester. For examples of preparations, see Bonnett, Nicolaidow, P. J, Chem.

Soc. Perkin Trans. I 1979, 1069-1077. Schaad, L.J., Werner, Dillon, Field, Tate, C.E. J. Med.

Chem. 1975, 18, 344-351, and Martin, T.A. J. Med. Chem.

1969, 12, 950-953.

Effects of compounds in a model of delayed type hypersensitivity in the mouse The property of the compounds diNAC, diBUT, diVAL, diCAP, diMeNAC, diEtNAC and diMeVAL to stimulate immune responses is illustrated by their efficacy in a model of the delayed type hypersensitivity (DTH) reaction in the mouse.

Both male and female Balb/c mice obtained from Bomholtsgaard (Denmark) and Charlie Rivers (England), were used at the weight of 18-20 gram. 4-ethoxymethylene-2-phenyloxazolone (OXA) was purchased from DBH (England) and served as an antigen in this test.

The mice were sensitized, Day 0, by epicutaneous application of 150 4l absolute ethanol-acetone solution containing 3% OXA on the shaved thorax and abdomen. Treatment with the L-form of diNAC, diMeNAC, diEtNAC, diMeVAL, or vehicle (phosphate buffer, pH was initiated by oral feeding immediately after sensitization and continued once daily to Day 6. Seven days (Day 6) after the sensitization both ears of all mice were challenged on both sides by topical application of ul 1% OXA dissolved in peanut oil. Ear thickness was measured prior to and 24 or 48 hours after challenge using an Oditest spring calliper. Challenges and measurements were performed under light pentobarbital anasthesia. The intensity of the DTH reactions was expressed according to the formula: T 24 4 ,-To unm units, where tO, t24 and t48 represent the ear thickness before and 24 or 48 hours after challenge, respectively, in an individual test The results were expressed as the mean .The level of significance between means of the groups was obtained by Student's two-tailed t-test. Tables 1 and 2 show the results from 24 and 48 hours measurements; respectively, from a representative experiment with the L-form of diNAC.

The results show that L-diNAC, after oral administration, caused a significant increase of the ear thickness in a concentration-response manner.

WO 91/18594 PCT/SE9I/00388 9 Table I.

Ear thickness 24 hours after challenge of animals treated with the indicated doses of L-diNAC or vehicle.

Conc.

jimnol kg Diff T,2 4 -T~r 0 S. E. M. Sign.

Buffer 13 7.85 0.32 NaCl 10 7.90 0.30 n.s.

0.03 10 13.75 0.47 0.30 10 15.70 0.48 10 18.30 1.02 30.0 15 20.67 0.67 p 0.001 WO 91/18594 PCT/SE91/00388 Table 2 Ear thickness 48 hours after challenge of animals treated with the indicated doses of L-diNAC or vehicle.

Conc.

imol/kg Diff T4aB-To S.E.M. Sign.

Buffer 14 9.64 0.35 NaCI 10 9.85 0.54 n.s.

0.03 10 11.65 0.27 0.30 10 12.65 0.48 10 14.95 0.55 30.0 15 13.63 0.30 p 0.001 Table 3 gives the correponding figures for ear thickness 24 and 48 hours after challenge of animals treated with diMeNAC and diEtNAC.

WO 91/18594 WO 9118594PC'/SE91/00388 Table 3 Ear thickness 24 and 48 hours after challenge of animals treated with the L-forms of dil~eNAC and diEtNAC.

Conc pnmol/kg N Dif f S. E.M. Sign.

24 h Buffer 10 8.70 diMeNAC 0 .03 0.03 18.00 12.55 11.75 13.05 0.34 0.84 0.88 0.62 0. 59 diEtNAC (to continue WO 91/18594 WO 918594PI'/SE9I /00388 table 3) Conc Wrno kg N Dif f S. E.M. Sign.

48 h diMeNAC diEtNAC 0.03 0 .03 12.85 13.35 13.15 13.20 0.67 0 .67 0.53 0.66 0.01 0.001 WO 91/18594 PCT/SE91/00388 13 Pharmaceutical formulations The described active substances can be included in different dosage forms e.g. tablets, coated tablets, gelatin capsules, solutions and aerosols.

For the preparation of tablets, coated tablets and gelatin capsules the active substances can be combined with pharmaceutically acceptable materials, e.g. lactose, starch, dicalcium phosphate, microcrystalline cellulose, polyvinylpyrrolidone, gelatin, cellulose derivatives, colloidal silicone dioxide, talc and stearic acid or its salts.

For the preparation of oral solutions suitable excipients are water, saccharose, glucose, sorbitol, fructose and xylitol.

The dosage forms can besides mentioned excipients contain preservatives, stabilizers, viscosity regulating agents, emulsifiers, sweetening agents, colouring agents, flavouring agents, tonicity regulating agents, buffers or antioxidants. They can also contain other therapeutically valuable substances.

WO 91/18594 PCT/SE91/00388 Example 1 Tablet containing 10 mg of active substance per tablet: Active substance Lactose Potato starch Polyvinylpyrrolidone Microcrystalline cellulose Magnesium stearate 10 mg 100 mg 50 mg 5 mg 15 mg 1 mg Example 2 Direct compression tablet containing 5 mg of active substance per tablet: Active substance Lactose, anhydrous Microcrystalline cellulose Colloidal silicon dioxide Magnesium stearate 5 mg 150 mg 50 mg 1 mg 2 mg If desired, the obtained tablets can be film coated with e.g. hydroxypropyl methylcellulose, hydroxypropyl cellulose or dimethylaminoethyl methacrylate methacrylic acid ester copolymer.

Example 3 Solution for injection containing active substance 1 mg/ml Active substance Sodium chloride Water for injection 1.0 mg 8.8 mg to 1 ml WO 91/18594 PCT/SE91/00388 Example 4 Oral solution containing active substance 1 mg/ml Active substance 1.0 mg Sorbitol 150 mg Glycerin 100 mg Disodium edetate 0.5 mg Preservative q.s.

Flavour q.s.

Water, purified to 1 ml Example Powder aerosol giving 1 mg per dose The micronized active substance can be filled into a powder inhaler device e.g. Turbuhaler giving 1 mg/dose.

Claims (13)

1. A method for the treatment of disease due to defects in the immune system, particularly chronic bronchitis, asthma, rhinitis, diabetes, rheumatoid arthritis, malignant diseases, HIV infection/AIDS and atherosclerotic disease, in mammals including man, characterized by the administration to a host in need of such treatment of an immunomodulating effective amount (particularly an immunostimulating amount) of racemic N,N'- diacetylcystine, or N,N'-dibutyrylcystine, or N,N'- diisovalerylcystine, or N,N'-dicaprylylcystine, or N,N'- diacetylcystine dimethylester, or N,N'-diacetylcystine diethylester, or N,N'-diisovalerylcystine dimethyl ester, or D and L optical isomers thereof, or a physiologically acceptable salt thereof, optionally together with a pharmaceutically acceptable carrier.

2. A method according to claim 1 wherein the L optical isomer of diacetylcystine, or dibutyrylcystine, or diisovalerylcystine, or N,N'-dicaprylylcystine, or N,N'-diacetylcystine dimethylester, or N,N'-diacetylcystine diethylester, or N,N'-diisovalerylcystine dimethyl ester, or a physiologically acceptable salt thereof, is used for the purpose.

3. A method according to claim 1 wherein N,N'- diacetyl-L-cystine is used for the purpose.

4. A method according to any one of claims 1 to 3 wherein the disease under treatment ie chronic bronchitis. A method according to any one of claims 1 to 3 wherein the disease under treatment is asthma.

6. A method according to any one of claims 1 to 3 wherein the disease under treatment is rhinitis. 1 I

7. A method according to any one of claims 1 to 3 wherein the disease under treatment is diabetes.

8. A method according to any one of claims 1 to 3 wherein the disease under treatment is rheumatoid arthritis.

9. A method according to any one of claims 1 to 3 wherein the disease under treatment is malignant. A method according to any one of claims 1 to 3 wherein the disease under treatment is HIV infection/AIDS.

11. A method according to any one of claims 1 to 3 wherein the'disease under treatment is atherosclerotic. DATED this 5th day of July, 1994 AKTIEBOLAGET ASTRA, By its Patent Attorneys, E. F. WELLINGTON CO., By: S. Wellington) A/RR/2966/9 INTERNATIONAL SEARCH REPORT International Application No PCT/SE 91/00388 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) 6 According to International Patent Classification (IPC) or to both National Classification and IPC A 61 K 31/195, 31/22, 31/225//C 07 C 323/59 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols A 61 K; C 07 C Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in Fields Searched 8 SE,DK,FI,NO classes as above III. DOCUMENTS CONSIDERED TO BE RELEVANT S Category Citation of Documentl with indication, where appropriate, of the relevant passages 2 Relevant to Claim No. 1 3 A EP, Al, 0300100 (MORGAN LEE ROY) 1-19 January 1989, see claims 1, 12, 19-24, 25, 32, 39-42; S p. 4, 1. 14-19 X 20-23 A US, A, 4827016 R. MORGAN) 2 May 1989, 1-19 see col. 5, 1. 49-67; col. 7, 1. 25-45; col. 10. 1. 34-61; col. 11, 1. 55 col. 12, 1. 9; col. 12, 1. 22-57 X 20-23 A US, A, 4724239 MORGAN) 9 February 1988, 1-19 see claims 1-4, 6-10, 12, 13, 15; col. 2, 1. 34-40 X 20-23 SSpecial categories of cited documents: 10 T' later document published alter the international iling date A dcrent d d efining, a the art which iity date and not in conflict with e application but eneral state of the art which is not cited to understand the principle or theory underlying the considered to be f pa icular relevance invention earlier document but published on or alter the international S lin b date 'X document of particular relevance, the claimed invention cannot be considered novel or cannot be considered to L* document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another citation or other special reason (as specified) document of particular relevance, the claimed invention cannot be considered to involve an inventive step when the document to an oral disclosure, use, exhibition or document is combined with one or more other such docu- "0 documen re(erring to an oral disclosure, use, exhibition or be g ors o'aheers d e other mearng merits, such combination being obvious to a person skilled other means in the art. document published prior to the international filing date but document member of the same patent family later than the priority date claimed& document member o the same patent faly IV. CERTIFICATION Date of the Actual Completion of the International Search Date ol Mailing of this International Search Report 28th August 1991 1991 -09- 1 1 International Searching Authority Signature of Authorized OffKer SWEDISH PATENT OFFICE Gerd Wranne Form PCT/ISAJ210 (second sheet) (January 1985) International Application No. PCT/SE 91/00388 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, rith indication, where appropriate, of the relevant passages Relevant to Claim No A US, A, 4708965 MORGAN) 24 November 1987, 1-19 see claims 1-3, 5; col. 5, 1. 50-67; col. 4, 1. 19-28 X

20-23 A Journal of Medicinal Chemistry, Vol. 18, No. 4, 1-19 1975 L.J. Schaad et al.: "Linear Regression Analysis of Inhibitory Potency of Organic Disulfides agains Histoplasma capsulatum", see p. 347, compound no. 72 X 20-23 A Journal of Medicinal Chemistry, Vol. 12, September 1-19 1969 T.A. Martin: "N-Acyl- and N-Sulfonylcysteine Derivatives", see page 950 page 953 see p. 952, Table I, compounds no. 19 and p. 950. right col.,last paragr aph p. 951, left col. first paragraph X 20-23 A FR, M, 8205 MORELLE ET AL.) 1-19 26 October 1970, see claims 1, 2; p. 2; first paragraph, 1. 28-31 X 20-23 A FR, Al, 2503151 MORELLE ET AL.) 1-19 8 October 1982, see p. 3; claims X 20-23 P,X International Journal of Pharmaceutics, Vol. 62, 1-23 1990 A.H. Kahns et al.: "Prodrugs as drug delivery systems. 107. Synthesis and chemical and enzymatic hydrolysis kinetics of various mono- and diester prodrugs of N-acetylcysteine", see page 193 page 205 see the whole article X Biochemical Pharmacology, Vol. 38, No. 22, 1989 K. 1-23 Sjodin et al.: "Metabolism of N-acetyl-L-cysteine. Some structural requirements for the deacetylation and consequences for the oral bioavailability", see page 3981 page 3985 see the whole article For PCT/ISA/2 tra s t) (Jan y 1985) Form PCT/ISA/210 (extra sheet) (January 1985) Internatonal Application No. PCT/SE 91/00388 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, with indication, where approiate, of the relevant passage Relevant to Claim No US, A, 3952115 DAMICO rT AL.) April 1976, see claims 1, 5, 6, 8-10; col. 5, 1. 17-30; col. 9, 1. 63 col. 10, 1, col. 10, 1. 68 col. 11, 1. 1 Tetrahedron Letters, Vol. 22, No. 16, 1981 W.R. Bowman et al.: "Reactions of thiolate anions with 2-substituted-2-nitropropanes", see page 1551 page 1554 see p. 1552, the Table, last compound DE, A, 2326444 (THE PROCTER GAMBLE CO.) 6 December 1973, see p. 7, 3:rd paragraph; p. 8; p. 2:nd paragraph; p. 21, last paragraph; p. 22, last paragraph p.-23, first paragraph Patent Abstracts of Japan, Vol 12, No 47, C475, abstract of JP 62-195356, publ 1987-08-28 SEIWA KASEI K.K. J. Org. Chem., Vol. 54, 1989 D.S. Kemp et al.: "Templates for Intramolecular O,N-Acyl Transfer via Cyclic Intermediates Derived from Mercury Derivatives of L-Cysteine: Progress toward a Mercury-Based Thiol Capture Strategy", see page 3853 page 3858 see p. 3855, right col. 2:nd paragraph 1-23 1-23 1-23 1-23 1-23 Form PCT/ISA/210 (extra sheet) (January 1985) International Application No. PCT/SE 91/00383 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V. S OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE This international search report has not been established in respect of certain claims under Article 17(2) for the following reasons: 1. Claim because 4 relate to subject matter not required to he searched by this Authority, namely Methods for treatment of the human or animal body by surgery or therapy, as well as diagnostic methods. /PCT Rule 39.1 2. Claim because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically. 3, Claim numbers because they are dependent claims and are not drafted in accordance with the second and third sen- tences-of PCT Rule 6.4(a). VI. E OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING This International Searching Authority found multiple inventions in this international application as lollows: 1. As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims of the international application. 2. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the international application for wnich fees were paid, specifically claims: i No required additional search fees were timely paid by the applicant. Consequently, this international search report is restrict- ed to the invention first mentioned in the the claims. It is covered by claim numbers: 4. As all searchable claims could be searched without ellort justifying an additional fee, the International Searching Authority did not invite payment of any additonal fee. Remark on Protest -i The additional search fees were accompanied by applicant's protest SNo protest accompanied the payment of additional seach fees. Form PCT/ISA/Z10 (supplemental sheat (January 1965) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO.PCT/SE 91/00388 This anntx lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The memLers are as contained in the Swedish Patent Office EDP rite on 91-06-27 The Swediih Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document cited in search report Publication date Patent family member(s) Publication date EP-Al- 0300100 89-01-25 US-A- 4708965 87-11-24 US-A- 4827016 89-05-02 US-A- 4827016 89-05-02 US-A- 4724239 88-02-09 EP-A- 0300100 89-01-25 US-A- 4708965 87-11-24 US-A- 4724239 88-02-09 US-A- 4827016 89-05-02 US-A- 4708965 87-11-24 EP-A- 0300100 89-01-25 US-A- 4827016 89-05-02 FR-M- 8205 70-10-26 NONE FR-Al- 2503151 82-10-08 DE-A-C- 3212448 82-11-11 GB-A-B- 2097256 82-11-03 JP-B- 2042805 90-09-26 JP-A- 57183703 82-11-12 US-A- 4859653 89-08-22 US-A- 3952115 76-04-20 AT-B- 348847 79-03-12 AU-B- 506795 80-01-24 AU-0- 1235476 77-09-29 BE-A- 840351 76-10-04 CA-A- 1064316 79-10-16 CH-A- 623206 81-05-29 DE-A-B-C 2613786 76-10-14 FR-A-B- 2305938 76-10-29 GB-A- 1533780 78-11-29 JP-A- 51142551 76-12-08 NL-A- 7603445 76-10-05 SE-B-C- 423301 82-05-03 SE-A- 7603892 76-10-03 DE-A- 2326444 73-12-06 AT-B- 339123 77-10-10 AU-B- 472310 76-05-20 AU-D- 5593173 74-11-21 BE-A- 800030 73-11-26 CA-A- 1004905 77-02-08 CH-A- 583003 76-12-31 DE-C- 2366239 82-04-01 FR-A-6- 2185360 74-01-04 GB-A- 1392151 75-04-30 JP-C- 826212 76-08-31 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO.PCT/SE 91/00388 This annex lists the patent family members relating to the patent documents cited In the obow..entlonditrainlsac eot The members are as contained in the Swedish Patent Orfice EDP fie on 9106-27 The Swedish Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent' document Publication Patent family Publication C cited in search report I date Imember~s) Idate DE-A- 2326444 73-12-06 JP-A- ~JP-B- NL-A- SE-B-C- US-A- 49124244 50037731 7307293 388113 3878305

74-11-28

75-12-04 73-11-27

76-09-27 75-04-15